The present invention relates to compositions and methods for use in reducing the adhesiveness of pathologically adhesive red blood cells (as hereinafter defined). In particular, the present invention is directed to compositions and methods for treatment of various conditions involving red blood cells with pathologically increased adhesiveness, for example as a result of malaria, sickle cell disease, thalassemia or diabetes.
This invention was made with Government support under Grant No. R01 AJ32995 and R01 DK16095, awarded by the National Institutes of Health. The Government has certain rights in this invention.
Sickle cell disease is a result of the presence of the altered gene product hemoglobin S. This disease is characterized by hemolytic anemia and complications resulting from episodic vaso-occlusive events, and despite the fact that more than 50 years have elapsed since the existence of a "vicious cycle" of sickling and erythrostasis was reported [Ham and Castle (1940) Trans. Assoc. Am. Physicians 55, 127-132], there still remain significant gaps in understanding of the mechanisms whereby sickle hemoglobin leads to the various manifestations of this disorder. Although the tendency of hemoglobin S to polymerize with reduced oxygen tension is the fundamental abnormality in sickle cell disease, polymerization and sickling itself do not entirely explain the pathophysiology of this disorder. In particular, membrane alterations in the sickle red cell contribute to sickle cell disease. The well-recognized complications of this syndrome such as recurrent and episodic painful crises, ischemic damage to tissues and organs, increased infections, and stroke presumably result from local disturbances in blood flow. The debilitating episodes of sickle cell crisis have been difficult to manage other than with hydration and analgesia.
Investigations have centered on the interaction of the sickle red cell and the vascular endothelial cell, in an attempt to identify those factors that could provoke a delay in microvascular flow. It was found that sickle red cells had a higher degree of adhesiveness to cultured human vascular endothelial cells than normal cells, and that this required neither frank morphologic deformation of the cells nor deoxygenation [Hoover et al. (1979) Blood 54, 872-876; Hebbel, R. P. et al. (1980) J. Clin. Invest. 65, 154-160]. This seminal observation with human umbilical vein endothelial cells was later confirmed both in static and flow systems using endothelial cells from a variety of tissues, and from mammalian sources other than humans. Significantly, among patients with sickle cell anemia, frequency of acute vaso-occlusive crises correlates with red blood cell adherence to endothelium. Accordingly, sickle cell adherence to endothelium was identified as the likely factor that initiates acute vaso-occlusion in sickle-cell disease, either by primarily occluding small vessels or by slowing microvascular blood flow so that secondary, reversible red blood cell sickling can occur.
Alterations in the surface of the sickle red cell involve changes in the distribution of surface charge, evidenced by the clustering of cationized ferritin on the surface of such cells; this was not found on cells containing normal hemoglobin [Hebbel & Eaton (1982) in Membranes and Genetic Disorders, A.R. Liss Inc., NY, pp. 311-349]. Calcium loading of normal cells induced both endothelial adherence and surface clumping of cationized ferritin. The binding of sickle cells might thus involve a redistribution of proteins. The mechanisms that might underlie such redistribution, however, were not revealed.
In the normal red cell, the integral proteins glycophorin and band 3 are randomly distributed in the membrane. Treatments of red cells to produce hemoglobin denaturation (hemichrome formation), ATP depletion, calcium loading, and oxidative cross-linking can all result in the formation of clusters of integral membrane proteins which may be visualized by freeze fracture electron microscopy. Clusters of intramembranous particles (composed principally of band 3 and glycophorin) are apparent at sites of brilliant cresyl blue induced hemichrome binding in .alpha.-thalassemic cells [Lessin, L. S. et al. (1972) Arch. Intern. Med. 129:306-319], in phenylhydrazine-treated cells [Low, P. S. (1989) in Hematology, Red Blood Cell Membranes: Structure, Function, Clinical Implications, Vol. 11, P. Agre & J. C. Parker, eds., Marcel Dekker, Inc., pp. 237-260], in erythrocytes that contain mature forms of P. falciparum [Allred, D. R. et al. (1986) J. Cell Science 81:1-16], and in irreversibly sickled cells [Lessin, L. S. et al. (1974) Proceedings of 1st Natl. Sympos. on Sickle Cell Disease, NIH, Bethesda, Md., pp. 213-214]. The processes that might drive such intramembrane clustering reactions in vivo have heretofore not been elucidated.
Sickle red cells generate excessive amounts of superoxide due to accelerated auto-oxidation of sickle heme [Hebbel et al. (1982) J. Clin. Inv. 70, 1253-1259]. This oxidant damage affects cellular hydration, increases hemichrome levels, causes the formation of hemichrome-stabilized membrane protein aggregates within the cell, and enhances adhesiveness. These same phenomena can be simulated in normal red cells by calcium loading or by treatment with the oxidant phenazine methosulfate [Hebbel et al. (1989) Am. J. Physiol. 256, C579-C583]. Further, free iron is non-randomly associated with the co-clusters of hemichrome and band 3 [Repka et al. (1993) Blood 82, 3204-3210], and could provide additional oxidant stress, focus damage to the underlying membrane structure, and promote further local hemichrome formation.
Adhesiveness is also observed in malaria-infected red cells. The hallmark of P. falciparum infections is sequestration, that is, attachment of erythrocytes infected with the mature stages of the parasite to the endothelial cells lining the post-capillary venules. This occurs principally in the lung, kidney, liver, heart and brain [Aikawa, M. et al. (1990) Am. J. Trop. Med. Hygiene 43:30-37; Pongponratn, E. et al. (1991) Am. J. Trop. Med. Hygiene 44:168-175]. Sequestration may totally occlude blood flow and result in tissue ischemia, coma, and death.
As with sickle and P. falciparum-infected erythrocytes the red blood cells from patients with diabetes have an abnormal adherence to the endothelium [Chappet, O. et al. (1994) Nouv. Rev. Fr. Hematol. 36: 281-288]. The mortality and morbidity from diabetes are related to the vascular complications resulting from vasoocclusion as well as capillary damage, and more than 75% of diabetic patients die from vascular complications. One of the consequences of the high concentrations of glucose in the blood plasma is the non-enzymatic glycosylation (glycation) of a variety of proteins such as those of the red cell membrane as well as hemoglobin. The early glycation products undergo a slow series of chemical rearrangements to form irreversible advanced glycation end products (AGE) and these accumulate over the lifetime of the proteins, including those of the erythrocyte. The AGEs are potentially pathogenic, and bind to receptors on the endothelium. Band 3 protein is easily accessible to glycation, and abnormal clustering of intramembranous particles has been shown for diabetic red cells [Rambini, R. et al. (1993) Membrane Biochemistry 10: 71-80]. Therefore, it is likely that alterations in the conformation of band 3, in concert with fibrinogen and to a lesser extent fibronectin, play a role in the enhanced adhesion of the red cell in diabetics.
The presence of sickle hemoglobin, hemoglobin S, is the underlying cause of sickle cell disease. The thalassemias are disorders of the red cell which involve a decreased synthesis of either of the protein chains of adult hemoglobin, hemoglobin A. This lack of coordination in synthesis leads to an accumulation of one chain relative to the other, and as a consequence the free chains aggregate and accumulate as insoluble inclusions at the inner face of the membrane, bound principally to band 3 protein. Erythrocytes from patients with thalassemia bind to endothelial cells to a greater degree than do normal red cells, and such patients have greater risk of vascular occlusion. Addition of autologous platelet-rich plasma causes a further increase in the number of adherent thalassemic red cells [Butthep, P. et al. (1992) S.E. Asian J. Trop. Med. and Public Hlth. 23, suppl 2, 101-104]. Thalassemic cells are enriched in calcium, the membranes of such cells when extracted with the non-ionic detergent Triton X-100 retain twice the amount of band 3 as does that of the normal red cell, and clusters of intramembranous particles (composed primarily of band 3 and glycophorin) are apparent at sites of brilliant cresyl blue induced hemichrome binding in thalassemic cells [Lessin, L. et al. (1972) Arch. Int. Med. 129:306-319]. Despite the fact that thalassemia and sickle cell disease are due to different genes, in both syndromes the red cell is similarly altered.
It is an object of the present invention to provide compositions and methods which are useful in reducing the adhesiveness of red blood cells which exhibit enhanced adhesive properties relative to normal red blood cells (e.g., sickle cells, malaria-infected red cells, thalassemic red cells, and red cells from diabetics).